It has been established that bone contains materials which can stimulate the formation of new bone when placed in contact with living systems. (Urist, M. R., Clin Orthop (1968) 56:37; Science (1965) 150:893; Reddi, A. H., et al., Proc Natl Acad Sci (USA) (1972) 69:1601.) Attempts have been made to purify whatever factors are responsible for this activity. A "bone morphogenetic protein" (BMP) was extracted from demineralized bone using urea or guanidine hydrochloride and reprecipitated according to the disclosures in U.S. Pat. Nos. 4,294,753 and 4,455,256 to Urist. Urist subsequently reported (Urist, M. R., Clin Orthop Rel Res (1982) 162:219) that ion exchange purification of this crude protein mixture yielded an activity which was unadsorbed to carboxymethyl cellulose resin (CMC) at pH 4.8. Urist's reports in Science (1983) 220:680-685 and Proc Natl Acad Science (USA) (1984) 81:371-375 describe BMPs having molecular weights of 17,500 and 18,500 daltons. Urist's patent publication, EPA Publication No. 0212474, describes BMP fragments of 4,000 to 7,000 daltons obtained by limited proteolysis of BMP.
U.S. Pat. No. 4,608,199 describes a bone-derived protein of 30,000-32,000 daltons. The protein is described as being water soluble and having no affinity for concanavalin A (ConA).
WO 88/00205 reports four proteins, designated BMP-1, BMP-2 Class I ("BMP-2"), BMP-3, and BMP-2 Class II ("BMP-4"), that are alleged to have osteogenic activity. It is not known whether these BMPs have osteogenic activity by themselves or require combination with other factors.
J. M. Wozney, in Growth Factor Research, Vol. 1 (1989), pp. 267-280, describes three additional BMP proteins closely related to BMP-2, and which have been designated BMP-5, BMP-6 and BMP-7.
WO 89/09787 and 89/09788 describe a protein called "OP-1" now known to be BMP-7. The cloning of BMP-7 is described in E. Ozkaynak et al., EMBO Journal (1990) 9:2085-2093, and the purification of BMP-7 is described in T. K. Sampath et al., J Biol Chem (1990) 265:13198-13205.
U.S. Pat. No. 4,434,094 to Seyedin and Thomas reported the partial purification of a bone generation-stimulating, bone-derived protein by extraction with chaotropic agents, fractionation on anion and cation exchange columns, and recovery of the activity from a fraction adsorbed to CMC at pH 4.8. This new protein fraction was termed "osteogenic factor" (OF) and was characterized as having a molecular weight below about 30,000 daltons.
Commonly owned U.S. Pat. No. 4,744,322 describes two bone-derived proteins that were purified to homogeneity by extraction with chaotropic agents, and ion exchange column fractionation. These two proteins were originally called cartilage-inducing factor (CIF) A and CIF B. CIF A was subsequently found to be identical to a previously identified protein called transforming growth factor beta (TGF-.beta.). CIF B has been found to be a novel form of TGF-.beta. and is now known as TGF-.beta.2, while CIF A is known as TGF-.beta.1.
Additional TGFs have also been described. U.S. Pat. No. 4,886,747 to Derynck et al., describes the identification of TGF-.beta.3 and its nucleotide sequence, and describes a method for recovery of TGF-.beta.3 from recombinant cell cultures. S. B. Jakowlew et al., Molec Endocrinol (1988) 2:1186-1195 describes TGF-.beta.4 and its nucleotide sequence, identified by cDNA characterization. A. B. Roberts et al., Growth Factors, Vol. 2 (1990), pp. 135-147, describes the purification of TGF-.beta.5 from Xenopus-conditioned medium.
A novel glycoprotein preparation from bovine bone designated osteoinductive factor (OIF), based on the ectopic osteoinductive activity used to follow its purification, has also been reported (Bentz, H., et al., J. Biol. Chem. (1989) 264:20805-20810). It was first thought that the osteoinductive activity of these "OIF preparations" could be substantially enhanced by either TGF-.beta.1 or 2 (Bentz, H., et al., J. Biol. Chem. (1989) 264:20805-20810; Bentz, H., et al., Development and Diseases Of Cartilage and Bone Matrix (A. Sen and T. Thornhill eds.) pp. 137-147, Alan R. Liss, Inc. (1987) New York). However, it was later found that the factors believed to have OIF activity did not in fact have that activity.
It is not known whether bone-inducing activity in isolated preparations is attributable to a single protein or a plurality of proteins acting in concert. Identification of the protein(s) responsible for bone-inducing activity is complicated by the large number of proteins extracted from bone (estimated to be several hundred), and the lack of a conclusive in vitro assay for bone-inducing activity.